This invention relates to substantially pure forms of cyclic boroproline compounds that bind, in cyclic or linear form, to CD26. The invention also relates to methods for using these compounds to stimulate the activation and/or proliferation of CD26-bearing cells to mobilize hematopoietic progenitor cells to spleen and periphery.
CD26, a type II transmembrane protein, is expressed on the cell surface of a number of cell types, including lymphocytes (Marguet, D. et al., Advances in Neuroimmunol. 3:209-215 (1993)), hematopoietic cells (Vivier, I. et al., J. Immunol. 147:447-454 (1991); Bristol, et al., J. Immunol. 149:367 (1992)), thymocytes (Dang, N. H. et al., J. Immunol. 147:2825-2832 (1991), Tanaka, T. et al., J. Immunol. 149:481-486 (1992), Darmoul, D. et al., J. Biol. Chem. 267:4824-4833 (1992)), intestinal brush border membrane, endothelial cells, fibroblasts, and stromal cells. Cell surface associated CD26 is a sialoglycoprotein, with most of its mass on the outside of the cell.
CD26 has been best characterized on peripheral T cells where it functions as a potent costimulatory signal for T cell activation. Its surface expression is up regulated upon T cell activation (Dong, R. P. et al., Cell 9:153-162 (1996), Torimoto, Y. et al., J. Immunol. 147:2514 (1991), Mittrucker, H-W. et al., Eur. J. Immunol. 25:295-297 (1995), Hafler, D. A. et , J. Immunol. 142:2590-2596 (1989), Dang, N. H. et al., J. Immunol. 144:409 (1990)). CD26 has also been identified in rodents as an important regulatory surface receptor in hematopoiesis and lymphoid development (Vivier, I. et al., J. Immunol. 147:447-454 (1991)). The primary structure of CD26 is highly conserved between species (Ogata, S. et al., J. Biol. Chem. 264:3596-3601(1998)). In humans, CD26 reportedly is involved in the regulation of thymocyte activation, differentiation and maturation (Dang, N. H. et al., J. Immunol. 147:2825-2832 (1991); Kameoka, J. et al., Blood 85:1132-1137 (1995)).
CD26 has an enzymatic activity that is identical to that of Dipeptidyl Peptidase IV (DPP-IV), a serine type exopeptidase with high substrate specificity. It cleaves N-terminal dipeptides from proteins if the penultimate amino acid is proline, or in some cases alanine (Fleischer, B. Immunol. Today 15:180 (1994)).
A class of low molecular weight synthetic monomeric molecules with high affinity for CD26 have previously been developed and characterized (G. R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991); W. G. Gutheil and W. W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). These molecules have been shown to be potent and specific synthetic inhibitors for CD26""s associated DP IV proteinase activity.
Representative monomeric structures of these transition-state-analog-based inhibitors, Xaa-boroPro, include Pro-boroPro, Ala-boroPro, Val-boroPro, and Lys-boroPro. BoroPro refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group [B(OH)2]. Pro-boroPro, the most thoroughly characterized of these inhibitors has a Ki of 16 picomolar pM) (W. G. Gutheil and W. W. Bachovchin. Separation of L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32, 8723-8731 (1993)). Val-boroPro has even a higher affinity, with a Ki of 1.6 PM (W. G. Gutheil and W. W. Bachovchin. Supra; R. J. Snow, et al. Studies on Proline boronic Acid Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic Species Containing a Bxe2x80x94N Bond, J. Am. Chem. Soc. 116, 10860-10869 (1994)). Thus, these Xaa-boroPro inhibitors are about 10+6 fold more potent than the next best known inhibitors.
U.S. Pat. Nos. 4,935,493 (Bachovchin ""493) and U.S. Pat. No. 5,462,928 (Bachovchin ""928), both of which are incorporated herein by reference, disclose protease inhibitors and transition state analogs (the ""493 patent) and methods for treating transplant rejection in a patient, arthritis, or systemic lupus erythematosis (SLE) by administering a potent inhibitor of the catalytic activity of soluble amino peptidase activity of dipeptidyl peptidase type IV (DP-IV; (G. R. Flentke, et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function, PNAS (USA) 88, 1556-1559 (1991)).
PCT published application WO 98/00439 (Multivalent Compounds for Crosslinking Receptors and Uses Thereof) reports that in aqueous solution at all pH values, a boroProline-type CD26 inhibitor exists as a slowly equilibrating mixture of two conformations: an open chain structure which is inhibitory (active species), and a cyclic structure which is non-inhibitory (inactive species). The open, active, inhibitory chain species is favored at low pH while the cyclized structure is favored at high pH. In view of the foregoing, the WO 98/00439 proposes preventing peptide conformational changes, e.g., intermolecular cyclization, by constructing a bivalent or multivalent compound containing an olefin group to form novel CD26 inhibitors. According to WO 98/00439, xe2x80x9cif cyclization can be blocked, the inventors predict that the bioavailability of the compounds taught herein can be increased by approximately 100-1000 foldxe2x80x9d.
The invention is based upon a variety of surprising and unexpected findings. It has been discovered, unexpectedly, that boro-Pro compounds of the type described in U.S. Pat. No. 4,935,493 (Bachovchin ""493) in cyclic form can be orally administered to a subject for treating the same types of conditions for which the linear molecules are useful. It is believed that the cyclic boro-Pro compounds undergo a transformation reaction under acidic conditions in vivo (e.g., stomach) to form a linear reaction product that is capable of selectively binding to CD26 (DP-IV). Thus, according to this aspect, the methods and compositions of the invention are directed to a novel pharmaceutical prodrug, namely, cyclic boro-Proline compounds, for oral administration. Novel compositions containing the substantially pure cyclic boroProline compounds of the invention, in solution or dry form, also are provided.
It is believed that the cyclic compounds of the invention are biologically active in cyclic form, as well as in linear form. Accordingly, the invention also embraces methods and compositions in which the cyclic compounds are administered to a subject or otherwise used in vitro (e.g., screening assays for selection of competitive molecules) in which the cyclic compound is not first subjected to conditions to induce conversion to the linear form. Thus, the cyclic compounds can be administered in oral form (whereby they may or may not be substantially converted to a linear form in the acidic conditions of the stomach), as well as in parenteral form with, or without, prior treatment to convert to the linear form.
The agents useful according to the invention are the cyclic forms of the compounds of Formula I (shown in FIG. 4), including all isomeric forms of this compound (discussed in more detail below. Referring to the Formula I compound, each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH; and X represents a side chain of an amino acid or a peptide which mimics the site of a substrate recognized by a post prolyl cleaving enzyme. In the preferred embodiments, the amino acids are L-amino acid residues (for glycine there is no such distinction); preferably, the C bonded to B is in the L-configuration. By xe2x80x9cthe C bonded to B is in the L-configurationxe2x80x9d is meant that the absolute configuration of the C is like that of an L-amino acid.
Peptides that mimic the substrate binding site of the post-prolyl cleaving enzyme DP IV (also referred to herein as xe2x80x9cCD 26xe2x80x9d) are described in U.S. Pat. No. 4,935,493 (xe2x80x9cBachovchin ""493xe2x80x9d) and U.S. Pat. No. 5,462,928 (xe2x80x9cBachovchin ""928xe2x80x9d).
The open chain (linear form) to cyclic form reaction involves a trans to cis isomerization of the proline and the formation of a new Nxe2x80x94B bond. Accordingly, xe2x80x9ccyclic formxe2x80x9d refers to the cyclized structure of the compounds of formula I that are the boron analogs of a diketopiperazine. This transformation is illustrated in FIG. 3.
By xe2x80x9csubstantially purexe2x80x9d it is meant that the cyclic compounds of the invention represent at least about 90% by weight of the composition. In the more preferred embodiments, the cyclic boroProline compound represents at least 98% by weight of the composition.
In certain embodiments, the cyclic boroproline compound represents a percentage by weight of the composition selected from the group consisting of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
In these and other embodiments, the compositions contain a linear boroProline compound that represents a percentage by weight of the composition selected from the group consisting of a percentage that is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.0%, and less than 0.5.
In a particularly preferred embodiment of the invention, the cyclic boroProline compound is a Val-boroProline compound. A xe2x80x9cVal-boroProline compoundxe2x80x9d refers to a compound of formula I in which the carboxy terminal boroProline is covalently coupled via a peptide linkage in accordance with standard peptide chemistry to a valine amino acid residue. A preferred embodiment is the cyclic form of Val-boroPro, which can be in the following forms: L-Val-S-boroPro, L-Val-R-boroPro, D-Val-S-boroPro, and D-Val-R-boroPro. More preferably, the compound is L-Val-S-boroPro or L-Val-R-boroPro.
According to yet another aspect of the invention, pharmaceutical compositions and methods for manufacturing such compositions are provided. The pharmaceutical compositions of the invention contain: (1) a pharmaceutically acceptable carrier; and (2) one or more of the cyclic boroProline compounds of the invention. Preferably, the pharmaceutical compositions are formulated for oral administration; however, lyophilized forms of the cyclic compounds are also provided for oral or parenteral administration. Such lyophilized or otherwise dried forms of the compounds of the invention may be reconstituted in a buffer of appropriate pH prior to administration. It is believed that oral formations containing the cyclic boroProline compounds of the invention undergo a conformation change from cyclic to linear form following administration, namely, when the compounds are subjected to the acidic pH conditions of the digestive system. For this reason, the preferred oral formations are tablets, capsules, or other solid forms which do not include an enteric coating. The method for manufacturing a pharmaceutical composition involves placing a cyclic boroProline compound of the invention in a pharmaceutically acceptable carrier and, optionally, formulating the cyclic compound into a tablet or other form that is suitable for oral administration.
According to another aspect of the invention, methods are provided for modulating immune system function. The compounds of the invention are administered to subjects in need of immune system modulation in amounts effective to modulate immune system function. Modulation of immune system function includes, but is not limited to, increasing immune function such as by stimulating proliferation and specific immune function of immune cells (e.g., CD26-bearing cells) to produce a prophylactic or therapeutic result relating to infectious disease, cancer, and the like. Specific conditions that may be treated according to the invention are deemed specific independent aspects of the invention and are described in detail in the examples. Exemplary conditions that can be treated by administering the compounds of the invention include: HIV infection; neoplasms (wherein the lymphocytes are cytolytic or helper T cells to attack the neoplasm); side effects of chemotherapy or radiation therapy (e.g., resulting from a depletion of cells of the immune system such as a depletion of cells derived from lymphoid, erythroid and/or myeloid lineages); kidney failure (e.g., resulting in depletion of cells of the immune system); bone marrow disorders resulting in immunodeficiency; autoimmunity; and immunodeficiency (e.g., resulting from depletion of cells of the immune system).
More particularly, the compounds of the invention are useful for the stimulation of proliferation, differentiation and mobilization of lymphocytes and hematopoietic cells, as well as for stimulation of cytokine production by stromal cells such as IL-6, IL-11, and G-CSF, and for stabilization or activation of cytokines which are substrates for CD26/DPP-IV protease activity (i.e., the cytokines terminate in the amino acid sequence XaaProline, wherein Xaa is an amino acid, and wherein the peptide sequence is subject to cleavage by CD26/DDP-IV). Accordingly, the invention is useful whenever it is desirable to stimulate the proliferation or differentiation of, or to mobilize, such immune cells, or to stimulate, stabilize or activate cytokine production. Mobilization of hematopoietic cells is characterized by the enrichment of early progenitor cells in the bone marrow and the recruitment of these cells to the periphery in response to a mobilization agent (e.g. G-CSF, GM-CSF, etc.). The agents useful according to the invention can be used to treat lymphocyte and hematopoietic cell deficiencies or to restore hematopoietic and mature blood cell count in subjects with such deficiencies. Such agents also may be used in connection with hematopoietic cell transplants, such as bone marrow or peripheral blood transplants, when used to replenish or create an immune system in a subject. The agents further can be used as an immune booster. The agents also are useful in vitro in connection with the culturing of cells for therapeutic and research uses.
The methods for stimulating activation or proliferation of human lymphocytes, hematopoietic cells, or stromal cells. The method involves contacting the lymphocytes, hematopoietic cells and/or stromal cells, in vivo or in vitro, with an activation or proliferation-inducing concentration of one or more cyclic boroproline compounds of the invention. In certain preferred embodiments, contacting is carried out by orally administering the compound to a human patient in need of such treatment, i.e., the patient is diagnosed as having an adverse medical condition characterized by inadequate lymphocyte or hematopoietic cell activation or concentration. Parenteral administration alternatively can be used to practice the method of treatment on a subject. As used herein, subject means humans, nonhuman primates, dogs, cats, sheep, goats, horses, cows, pigs and rodents.
The compounds of the invention can be administered alone, or in combination with additional agents for treating the condition, e.g., a different agent which stimulates activation or proliferation of said lymphocytes, hematopoietic cells and/or stromal cells. Contacting the lymphocytes with the compounds of the invention can be performed in vitro or in vivo.
As used herein, compound of the invention means the compounds described above as well as salts thereof.
These and other aspects of the invention will be described in greater detail below.
All patents, patent applications, references and other documents that are identified in this patent application are incorporated in their entirety herein by reference.
By xe2x80x9camino acidxe2x80x9d is meant to include imino acid.
By xe2x80x9cboroProxe2x80x9d is meant an alpha-amino boronic acid analog of proline bonded to an amino acid to form a dipeptide with boroPro as the C-terminal residue. xe2x80x9cBoroProxe2x80x9d is used to designate such an analog having the carboxyl group of proline replaced with a B(OH)2 group, where (OH)2 represents two hydroxyl groups and B represents boron.
By Xaa is meant any amino acid residue, e.g., a lysine residue, a valine residue.
xe2x80x9cCD26 ligandxe2x80x9d is any protein, glycoprotein, lipoprotein or polypeptide that binds to the T cell receptor CD26 and may provide a stimulatory or inhibitory signal.
CD26, Dipeptidyl Peptidase IV (DP IV or DPPIV) and dipeptidyl aminopeptidase IV are used interchangeably. CD26 is a postproline cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of polypeptides.
By alpha-carbon of an amino acid is the one to which the carboxylic acid group is attached. All naturally occurring amino acids are alpha-amino acids or alpha-imino, which means that the amino and carboxylic acid groups are both attached to the same carbon atom.
Each amino acid can be thought of as a single carbon atom (the alpha carbon, C) to which there is attached one carboxyl group, one amino group, a side chain denoted R and a hydrogen, wherein: xe2x80x9cRxe2x80x9d is a side chain; xe2x80x9cNH2xe2x80x9d is the alpha amino group; the first carbon (C) attached to the NH2 group having a hydrogen (H) and an R group attached is the alpha carbon; and the carbon double bonded to an oxygen and a hydroxyl group (OH) is the alpha carboxyl group.
The NH2 and COOH groups are used to covalently couple amino acids to one another. The hydroxyl group (OH) of one amino acid on the carboxyl end and the hydrogen (H) on the N terminus are removed (H2O) when two amino acids are linked together. To form a protein, the amino group of one amino acid reacts with the carboxyl group of another by the elimination of water; the resulting chemical bond is called a peptide bond.
By xe2x80x9cpeptidesxe2x80x9d is meant a small molecule, e.g., usually containing less than 50 amino acid residues, which do not generally possess a well-defined three-dimensional structure.
Pharmaceutical preparations and modes of administration are described herein.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.